Synthesis, molecular docking and DFT analysis of novel bis-Schiff base derivatives with thiobarbituric acid for α-glucosidase inhibition assessment

A library of novel bis-Schiff base derivatives based on thiobarbituric acid has been effectively synthesized by multi-step reactions as part of our ongoing pursuit of novel anti-diabetic agents. All these derivatives were subjected to in vitro α-glucosidase inhibitory potential testing after structural confirmation by modern spectroscopic techniques. Among them, compound 8 (IC50 = 0.10 ± 0.05 µM), and 9 (IC50 = 0.13 ± 0.03 µM) exhibited promising inhibitory activity better than the standard drug acarbose (IC50 = 0.27 ± 0.04 µM). Similarly, derivatives (5, 6, 7, 10 and 4) showed significant to good inhibitory activity in the range of IC50 values from 0.32 ± 0.03 to 0.52 ± 0.02 µM. These derivatives were docked with the target protein to elucidate their binding affinities and key interactions, providing additional insights into their inhibitory mechanisms. The chemical nature of these compounds were reveal by performing the density functional theory (DFT) calculation using hybrid B3LYP functional with 6-311++G(d,p) basis set. The presence of intramolecular H-bonding was explored by DFT-d3 and reduced density gradient (RGD) analysis. Furthermore, various reactivity parameters were explored by performing TD-DFT at CAM-B3LYP/6-311++G(d,p) method.

In continuous efforts to discover anti-diabetic agents our group has successfully synthesized a library of novel bis-Schiff base derivatives based on thiobarbituric acid through multi step reactions.In the first step, 2,4-dihydroxybenzaldehyde was refluxed with 1,3-diethyl-2-thiobarbituric acid in ethanol solvent containing catalytic amount of triethylamine for 2 h with constant stirring to get compound 1.In the second step, compound 1 was further refluxed with ethyl chloroacetate in DMF solvent having potassium carbonate for 3-4 h to obtain compound 2, which was further treated with excess of hydrazine hydrate in ethanol solvent to get the bis-hydrazide 3 in better yield.Finally, a number of aromatic aldehydes were refluxed with compound 3 for 4-5 h in absolute ethanol having catalytic amount of acetic acid to obtain bis-Schiff bases (4-10) (Fig. 2).Thin layer chromatographic technique was used to know the formation of products in solvent system of n-hexane and ethyl acetate (7:3).After the reactions completions, these mixtures were decanted to a beaker having cold distilled water.The appeared colored precipitates were filtered, washed with hot water followed by n-hexane, recrystallized with absolute ethanol to get the products in pure form.All the product compounds were structurally deduced through HR-ESI-MS, 13 C-, and 1 H-NMR spectroscopy.
The 1 H-NMR spectra of the synthesized compounds (4-10) revealed signals for several methine (-CH), methylene (-CH 2 ), methyl (-CH 3 ), and NH protons.In the down field region of the spectrum, singlet signals of two proton integrations appeared at 12.88-11.79were due to the -NH protons.Similarly, another singlet signals resonated in the range 9.93-7.98 was assigned to the methine (=CH) protons.In the spectrum, signals of the aromatic protons were seen at 8.39-6.98 and 5.54-5.30were due to the methylene protons of barbituric acid, while signals observed at 3.78-3.75were assigned to the methyl protons.The 13 C-NMR (DEPT and Broad Band) spectra of these derivatives exhibited signals for methyl, methylene, methine, and quaternary carbon atoms.The signal observed between 170.5 and 169.9 in the down field portion of the spectrum was caused by the carbonyl carbon.The signals at 165.2-159.9were created in the same way by a quaternary carbon containing oxygen and nitrogen atoms.The signal of the methylene group, on the other hand, resonates at 66.6-66.3.Furthermore, molar masses of these analogues were confirmed with the help of HR-ESI-MS spectra showing the molecular ion peaks.

Structure activity relationship study
It's important to note that all compounds were promising α-glucosidase inhibitors, demonstrating that each structural component contributes to the inhibitory activity.Since the aceto-hydrazide moiety, pyrimidine, and benzene ring are identical in all of the compounds, the limited structure-activity relationship (SAR) study is justified by the presence of distinct characteristics, such as various patterns of aryl ring substitution.In the synthesized derivatives, compound 8 (IC 50 = 0.10 ± 0.05 µM) was the most potent inhibitor of α-glucosidase enzyme.The potency of this compound could be due to the presence of electron withdrawing chlorine atoms attached to the benzene ring at ortho and para position.A very slight decline occurs in the activity of compound 9 (IC 50 = 0.13 ± 0.03 µM) could be due to the change of one chlorine atom from para to meta position of the benzene ring.Similar to this, the less activity of compound 10 (IC 50 = 0.42 ± 0.07 µM) might be due to the removal of one chlorine atom from the ortho position of the benzene ring from compound 9 (Fig. 3).By comparing compounds 5 (IC 50 = 0.32 ± 0.03 µM) with 6 (IC 50 = 0.36 ± 0.04 µM) and 7 (IC 50 = 0.39 ± 0.08 µM), the highest activity of compound 5 could be due to the attachment of nitro group at ortho position.Furthermore, change of position from ortho to para and meta position of the nitro group may be responsible in decreasing the activities of compounds 6 and 7 respectively.However, compound 4 (IC 50 = 0.52 ± 0.02 µM) showed less inhibitory activity among the series due to the attachment of electron donating hydroxyl group at para position of the benzene ring.Our structure activity relationship study showed that compounds having electron-withdrawing substituents displayed superior inhibitory activities.This correlation underscores the significance of these substituents in enhancing the potency of the product compounds.Moving forward, these insights provide a valuable foundation for optimizing future drug candidates in this compounds library.

NMR analysis
The NMR calculations were performed both experimentally and theoretically.The theoretical chemical shift values were simulated downfield from TMS at B3LYP/6-31+G(2d,p) method.The 1 H-NMR spectrum reveals distinctive chemical shifts for various functional groups.Notably, in the -CH= group, the experimental and calculated values are reasonably congruent, with a subtle variation observed.This suggests that the theoretical calculations effectively capture the electronic environment of functional groups.The most downfield chemical shift value was found for this group from 11.89 to 9.90 ppm.In contrast, the simulated 1 H-NMR peak for this group was found the downfield from 10.9 to 9.90 ppm range.The DFT and RGD analysis reveals the presence of intramolecular hydrogen bonding which makes the chemical shift downfield.Similarly, the -CH=N-group exhibit the 1 H-NMR peak from 10.9 to 9.90 ppm range while the calculated chemical shifts values were found downfield from 9.35 to 9.30 ppm.The -CH 2 group also display noteworthy agreement between the experimental and theoretical values.The experimental value for -CH 2 was found from 5.10 to 4.28 ppm downfield from TMS, whereas, the calculated values observed from 4.84 to 4.60 ppm.The chemical environment of aromatic protons is different due to the presence of heteroatoms within the ring.Due to the presence of heteroatoms 1 H-NMR chemical shift values were observed form 8.31-6.14ppm experimentally, while the calculated values were found from 8.8 to 6.33 ppm.The detailed 1 H-NMR chemical shift values are listed in Table 2.
Turning to the 13 C-NMR spectrum, the experimental values for carbon atoms within the 168.3-39.0ppm range.However, the calculated chemical shift values were observed within 190.0-49.60 ppm.In summary, the comparison between experimental and calculated chemical shift values across different functional groups in both 1 H-and 13 C-NMR spectra suggests that the DFT calculations provide a reliable approximation of the molecular structure and electronic characteristics of the studied compounds.

RGD analysis
To know about the intermolecular/intramolecular interaction, the RGD analysis is a useful technique 41 .The RGD scatter plot provides the presence of different types of interactions i.e., hydrogen bonding, van der Waals (vdW) and steric effect in the synthesized compounds as shown in Fig. 5a.The scatter plot and 3D isosurfaces indicates the nature of non-covalent interactions associated with the values of sign (λ 2 )ρ values.
On the 2D scatter plot the sign (λ 2 )ρ value is associated with different types of interactions.The deep blue color on the scatter plot signifies a noticeably negative value of (λ 2 )ρ, indicating the presence of attractive forces such as hydrogen bonding or dipole-dipole interactions.In contrast, when observing the scatter graph, a positive (λ 2 )ρ value shows as a red color, pointing to the presence of repulsive forces.Conversely, a value close to zero but remaining negative is as indicated by a green color, indicating the presence of van der Waals interactions.On the scatter plot an observed positive peak range from 0.01 to 0.05 au in the red region signifies the presence of repulsive interaction which is due to steric effect.Furthermore, the range from − 0.01 to 0.01 au on the scatter plot indicates the presence of vdW interactions.The most important peak found in the RGD analysis is the peak that's range from − 0.02 to − 0.05 au.This peak signifies the presence of strong attractive interaction such hydrogen bonding as found in the synthesized compounds.Figure 5 provides the valuable insights into the nature of attractive, repulsive forces, and specific interactions such as hydrogen bonding and van der Waals forces within the synthesized compounds.

Frontier molecular orbital (FMO) analysis
For the FMO analysis, TD-DFT calculations were performed using the CAM-B3LYP functional with a 6-311++G(d,p) basis set.The FMO is useful technique used to know about the chemical nature of newly synthesized compounds.In FMO analysis, the HOMO and LUMO energies play a crucial role.The energy gap between these two states (ΔE gap ) was calculated as follows: These energy levels provide valuable information about electron distribution, leading to the determination of various properties as listed in Table 3.The energy difference (ΔE gap ) between HOMO and LUMO is a key factor for understanding a compound's chemical behavior.Compounds with larger energy gaps are less reactive and more stable because they need more energy for electronic excitation.On the other hand, compounds with   In the FMO calculations, several reactivity parameters were determined such as, ionization energy (IP), electron affinity (EA), electronegativity (x), chemical hardness (η) and softness (σ).The ionization energy gives the information about the removal of an electron from a compound and is linked to E HOMO .Ionization energy for the synthesized compounds ranged from 0.276 to 0.299.The compound 4 shows the lowest ionization energy while the compound 6 shows the highest ionization energy of 0.299.The lowest ionization energy of compound 4 suggests easier electron removal compared to other compounds.
The electron affinity (EA) is another important parameter in the chemical reactivity.The electron affinity is the reverse of ionization energy and is define as the amount of energy released by addition of an electron to compound.In this work, the calculated electron affinity values were noted from 0.092 to 0.101.The compound 4 indicated low amount of energy released by the addition of an electron, while reverse is observed for compound 6, which indicate the highest amount of energy release at 0.101.
Additionally, the molecular polarizability can be determined through softness and hardness.A molecule exhibits increased polarizability when it possesses a higher softness value along with a lower hardness value.Among the synthesized compounds, 4 is more polarizable due to its low hardness and high softness values.Detailed FMO parameters are listed in Table 3. Overall, the FMO analysis not only explained the electronic structure and reactivity of the synthesized compounds but also provided a comprehensive understanding of their chemical nature.

Molecular electrostatic potential (MEP) analysis
To identify the electrophilic and nucleophilic sites of synthesized compounds, the MEP analysis was performed at TD-DFT method.The MEP analysis is associated with charged particles within a compound.The blue color in the MEP Fig. 7 indicate the electron deficient region and is considered is a favorable position for nucleophilic attack.Conversely, the regions in red indicate higher electron density, signifying a higher concentration of electrons.These areas are regarded as the preferred sites for electrophilic attacks.

Molecular docking
The molecular docking of synthesized compounds with α-glucosidase was performed using the Auto Dock Vina package.The docking study helps to find the binding modes of synthesized compounds (ligands) with amino acids of protein.The synthesized compounds show different interaction with α-glucosidase.The protein structure of α-glucosidase was taken from Protein Data Bank (PDB) at (https:// www.rcsb.org/ struc ture/ 3WY1) with PDB Entry-3WY1.
In simulation, the water molecules were excluded, and any absent hydrogen atoms in the protein were added using Autodock Tools.The PDB structures of the synthesized compounds (ligands) were formed in pdb format based on the Gaussian optimized structures.The cubic grid box was made with dimensions of 3.391, 1.310, and − 8.362 Å in the x, y, and z-directions respectively.The grid box was centered on the middle of targeted compounds under docking.Grid maps were generated using the AutoGrid Tools with a grid spacing of 0.375 Å.The number of grid points in each direction (x, y, and z) was taken as 40.Additionally, PyMOL 2.5.4 was employed to visualize the interactions between the protein and ligands, while Biovia Discovery Studio was utilized for the 2D representation of protein-ligand interactions, as shown in Fig. 8.The simulation reveals different types of interactions between amino acid and ligands, such as conventional H-bonding, van der Waals, unfavorable positive-positive, attractive and π-interactions as shown in Table 4.
In conclusion, our docking study has provided a comprehensive insight into the binding affinities and molecular interactions of compounds 4-10 with the target protein.Notable interactions, including conventional hydrogen bonding, van der Waals interactions, and attractive charges/π-cation interactions, contribute to the overall binding profiles.These findings contribute valuable information for further optimization and refinement of these compounds for enhanced binding efficacy and potential therapeutic applications.

General
All of the chemicals, reagents and solvents utilized in this study were of synthetic grade and purchased from BDH, Sigma-Aldrich, and Merck.Using the solvent system of n-hexane and ethyl acetate, thin layer chromatography (TLC) has been carried out on pre-coated silica gel 60 F254 aluminium cards.To assess the melting points of the synthesized compounds, Stuart SMP10 melting point apparatus was used.The masses of these analogues were determined using advanced high resolution electrospray ionization mass spectrometry (HR-ESI-MS) (Agilent 6530 LC Q-TOF, USA/EU, produced in Singapore).On a Bruker Avance 150 MHz and 600 MHz spectrophotometer (BRUKER, Zürich, Switzerland), the 13 C-NMR and 1 H-NMR spectra were captured using solvent peaks as internal references (DMSO-d 6 , δ H : 3.34; δ C : 39.9-39.1)respectively.The abbreviations s: singlet, d: doublet, t: triplet, m: multiplet, J: coupling constant (in Hz), and for chemical shifts in parts per million (ppm) are used in the explanation of NMR spectra.In the first step, 1,3-diethyl-2-thiobarbituric acid was refluxed with 2,4-dihydroxybenzaldehyde in a 100 ml round bottomed (RB) flask in the presence of catalytic amount of triethylamine (Et 3 N) in 100% ethanol for 2 h to obtain compound 1, which was further refluxed for 3-4 h in DMF solvent with ethyl chloroacetate having catalytic amount of potassium carbonate (K 2 CO 3 ) to get the desired compound 2.Then, hydrazine hydrate was refluxed with compound 2 in ethanol to get the desired bis-hydrazide 3.At last, different substituted aromatic aldehydes were refluxed for 4-5 h with compound 3 in ethanol solvent having 4-5 drops of acetic acid to get the desired bis-Schiff bases of thiobarbituric acid nucleus.Thin layer chromatographic technique was established to check the formation of product compounds using 30% polar system of n-hexane and ethyl acetate (7:3).After formation of the products, colored precipitates were appeared by pouring the mixtures to cold distilled water; the precipitates were washed with distilled water after filtration and kept overnight for drying.These product compounds were recrystallized with absolute ethanol to get them in pure form.Structures of all the compounds were deduced through HR-ESI-MS, 13 C-, and 1 H-NMR spectroscopic techniques.

Computational details
All the density functional theory (DFT) calculations were carried out at Gaussian 09 package.The geometry optimizations were performed using a hybrid functional B3LYP with 6-311++G(d,p) basis set 42,43 .To further ensure the true minima, the frequency calculations were performed without imaginary frequency.For the account of intramolecular interaction the DFT-D3 approach was utilized.The NMR calculations were performed by Gauge-Invariant Atomic Orbital (GIAO) method at B3LYP/6-311+G(2d,p) 44,45 .The detailed electronic structural analysis was performed at TD-DFT using CAM-B3LYP/6-311++G(d,p) method.For the prediction of electrophilic and nucleophilic sites of synthesized compounds the molecular electrostatic potential (MEP) was performed.Furthermore, docking analysis was done by using AutoDock Vina 46 to investigates the binding between amino acids residues and protein (α-glucosidase).For docking analysis, the dimension of grid box was selected as 40, 40, and 40 in x, y, and z-direction with grid-point spacing of 0.375 Å.

Conclusions
In conclusions, we have reported impressive novel α-glucosidase inhibitors through the systematic synthesis of bis-Schiff base derivatives based on thiobarbituric acid scaffold.These derivatives were structurally confirmed through different spectroscopic techniques including HR-ESI-MS, 1 H-, and 13 C-NMR and finally screened them for their in vitro α-glucosidase inhibitory activity.In the series, compounds 8 and 9 displayed superior efficacy, better than the reference drug acarbose.These results highlight the possibility of thiobarbituric acid derivatives as effective candidates for the development of anti-diabetic medications.To move these derivatives closer to clinical use and support current attempts to find safe and effective diabetes therapies more research into their pharmacokinetics and safety profiles is necessary.Additionally, we have performed the docking study to elucidate the molecular interactions between these compounds and the target protein, offering a deeper understanding of their inhibitory mechanisms. Vol

Figure 6 .
Figure 6.The HOMO and LUMO energies with energy gap between the two states are presented in red color in (eV).

Table 2 .
Calculated and experimental NMR chemical shift values.

Table 3 .
Chemical reactivity parameters obtained from TD-DFT analysis.

Table 4 .
Representation of protein-ligand interactions.